Quantifying Immune Cell Infiltration in Uropathogenic E. coli-Infected Mouse Bladder Tissue using Flow Cytometry
Transcrição
To quantify immune cell infiltration in uropathogenic E. coli-infected mouse bladder tissue, place the minced tissue in a digestion buffer containing collagenase and deoxyribonuclease.
Incubate with regular shaking to disrupt the tissue.
Collagenase digests the tissue's extracellular matrix, releasing cells. Deoxyribonuclease degrades interfering free DNA.
Post-incubation, add buffer with a chelating agent and fetal bovine serum to inactivate the digestive enzymes.
Pass the digested mixture through a strainer to remove tissue debris and connective tissue, obtaining a single-cell suspension.
Centrifuge and resuspend the cells in a buffer containing Fc-blocking antibodies. These antibodies bind to immune cell Fc receptors to prevent non-specific antibody binding.
Add fluorophore-tagged anti-CD45 antibodies, which bind to the transmembrane CD45 protein on immune cells.
Centrifuge and pass the resuspended cells through a cell strainer to remove cell aggregates.
Using a flow cytometer, measure the fluorescence signals from the fluorophore-conjugated antibodies on immune cells to quantify immune cell infiltration in the bladder.
